U.S. patent application number 10/394998 was filed with the patent office on 2004-09-23 for adaptor for dye terminator removal apparatus.
This patent application is currently assigned to BIO-RAD LABORATORIES, INC.. Invention is credited to Aschettino, Danielle, He, Xuemei M., Hey, Julie S., Perry, Brian A., Seggerson, Carolyn M..
Application Number | 20040184968 10/394998 |
Document ID | / |
Family ID | 32988517 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040184968 |
Kind Code |
A1 |
Aschettino, Danielle ; et
al. |
September 23, 2004 |
Adaptor for dye terminator removal apparatus
Abstract
A gel filtration plate designed to remove unincorporated dye
terminator from a series of reaction mixtures in a nucleic acid
sequencing operation is joined to an injection plate designed for
insertion into a sequencing apparatus through an adaptor assembly
that allows the two plates to be centrifuged together, thereby
simultaneously purifying the reaction mixtures and collecting them
in the receptacles that will be placed directly in the sequencer.
The adaptor avoids the use of an intermediary collection plate and
the need to transfer reaction mixtures from each well of the
collection plate to each well of the injection plate.
Inventors: |
Aschettino, Danielle;
(Rodeo, CA) ; Perry, Brian A.; (Fremont, CA)
; Hey, Julie S.; (Fairfield, CA) ; He, Xuemei
M.; (Walnut Creek, CA) ; Seggerson, Carolyn M.;
(Alameda, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
BIO-RAD LABORATORIES, INC.
1000 Alfred Nobel Drive
Hercules
CA
94547
|
Family ID: |
32988517 |
Appl. No.: |
10/394998 |
Filed: |
March 21, 2003 |
Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01L 2200/025 20130101;
B01L 9/523 20130101; B01L 2300/0829 20130101; B01L 3/50255
20130101; B01L 2400/049 20130101; B01L 2400/0409 20130101 |
Class at
Publication: |
422/103 ;
422/101 |
International
Class: |
B01L 011/00 |
Claims
What is claimed is:
1. An adaptor assembly for joining a gel filtration plate having a
multitude of gel filtration wells arranged in a selected array and
an injection plate having a multitude of receptacles arranged in an
array identical to that of said gel filtration wells, said
injection plate being designed for direct insertion in an automated
nucleic acid sequencing apparatus, said adaptor assembly allowing
said gel filtration and injection plates to be centrifuged together
to simultaneously (i) purify product mixtures of a multitude of
nucleic acid sequencing reactions by removal of unincorporated dye
terminators from said product mixtures and (ii) collect said
product mixtures thus purified in said injection plate, said
adaptor assembly comprising: a support base to support said
injection plate, said support base having a peripheral wall of
sufficient height to fully cover the external surfaces of said
receptacles, and a bracket shaped to secure said gel filtration
plate, said injection plate, and said support base together while
contacting at least two opposing peripheral edges of said gel
filtration plate and said injection plate to maintain alignment
between each well of said gel filtration plate and a corresponding
receptacle of said injection plate.
2. An adaptor assembly in accordance with claim 1 in which said
support base comprises a plurality of apertures arranged in an
array identical to the array of said gel filtration wells and said
receptacles, each of said apertures of sufficient size to receive
one of said receptacles.
3. An adaptor assembly in accordance with claim 1 in which said
bracket comprises a base plate and a pair of parallel tabs
extending perpendicularly from opposing edges of said base
plate.
4. An adaptor assembly in accordance with claim 3 in which said
base plate is rectangular in shape and said bracket further
comprises a raised rim on all four edges of said base plate such
that said support base fits within said rim.
5. An adaptor assembly in accordance with claim 3 in which each of
said tabs contains an opening and said adaptor assembly further
comprises a U-shaped handle terminating at both ends in hooks that
engage said openings.
6. A gel filtration and adaptor combination for receiving an
injection plate which has a multitude of receptacles in a selected
array and is designed for direct insertion in an automated nucleic
acid sequencing apparatus, such that said combination together with
said injection plate forms an assembly that can be placed on a
centrifuge to simultaneously purify product mixtures from a
multitude of nucleic acid sequencing reactions by removal of
unincorporated dye terminators from said product mixtures and
collect said product mixtures thus purified in said injection
plate, said combination comprising: a gel filtration plate having a
multitude of gel filtration wells arranged in an array identical to
that of said injection plate, a support base shaped to permit said
injection plate to rest thereon, said support base having a
peripheral wall of sufficient height to fully cover the external
surfaces of all receptacles of said injection plate, and a bracket
shaped to secure said gel filtration plate, said injection plate,
and said support base together while contacting at least two
opposing peripheral edges of said gel filtration plate and said
injection plate to force each well of said gel filtration plate
into alignment with a corresponding receptacle of said injection
plate.
7. A gel filtration and adaptor combination in accordance with
claim 6 in which said bracket comprises a base plate and a pair of
parallel tabs extending perpendicularly from opposing edges of said
base plate, each tab having an opening to receive a hook, and said
gel filtration and adaptor combination further comprises a U-shaped
handle terminating at both ends in hooks that engage said
openings.
8. A gel filtration and adaptor combination in accordance with
claim 6 in which said base plate is rectangular in shape and said
bracket further comprises a raised rim on all four edges of said
base plate such that said support base fits within said rim.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention lies in the field of nucleic acid sequencing,
and addresses in particular the types of apparatus used for
preparing sequencing samples for analysis on automated
instrumentation.
[0003] 2. Description of the Prior Art
[0004] The determination of nucleotide sequences in genes and
chromosomes is typically done by DNA sequencing involving the
well-known polymerase chain reaction (PCR) with the use of dye
terminators, followed by electrophoresis of the reaction product
mixtures. The simultaneous electrophoresis of multiple product
mixtures and the compilation of the sequencing data from the
electropherograms is performed by automated analyzers that draw the
mixtures from injection plates and place the mixtures in the
capillaries where the electrophoretic separations occur, the
various transfers occurring by electrokinetic means. In order for
these separations to provide accurate and reliable sequencing data,
the reaction product mixtures must be purified prior to the
electrophoresis to remove unincorporated dye terminators and other
impurities that are either left unreacted or generated during the
reactions.
[0005] A common method of purifying the reaction product mixtures
is gel filtration assisted either by a vacuum or by centrifugation.
Gel filtration matrices and plates that contain arrays of
filtration wells are commercially available from suppliers of
sequencers. The typical filtration plate contains 96 wells in a
standard array with the filtration material forming a bed in each
well. Sequencing reaction samples are transferred by pipet to each
well, and the filtration plate is fitted over a collection plate
that has an array of wells corresponding to those of the filtration
plate. Thus fitted, the two plates are placed on a centrifuge.
After centrifuging, the collection plate is separated from the
filtration plate, and the purified samples are transferred from the
wells of the collection plate to the wells of an injection plate
that is inserted into the sequencer for automated handling and
electrophoresis. The injection plate is an array of receptacles
that hold the samples in the sequencer until automated syringes in
the sequencer draw the samples from the receptacles and transfer
the drawn samples to capillaries in which the electrophoresis
occurs.
[0006] The use of separate collection and injection plates and the
need to transfer the samples from one of these plates to the other
arises from the fact that the collection plate is designed for
placement in a centrifuge with the filtration plate while the
injection plate is designed for insertion into the sequencer, the
centrifuge and the sequencer being separate pieces of equipment.
Injection plates vary in construction depending on the particular
sequencer for which they are designed. In general, however, the
plates are simply supports for the receptacles whose closed ends
are exposed at the bottom. Injection plates from certain
manufacturers contain a skirt that surrounds the closed ends of the
receptacles although the skirts are in some cases designed as a
supporting feature rather than a protective feature. The skirt may
thus extend sufficiently far down to cover the entire length of the
receptacle, or it may extend only part way down leaving a portion
of the length of each receptacle exposed. Other injection plates
contain no skirt at all.
SUMMARY OF THE INVENTION
[0007] This invention resides in an adaptor assembly that permits a
multi-well gel filtration plate to be secured directly to a
multi-well injection plate so that both can be placed on a
centrifuge. The PCR reaction product mixtures can thus be purified
and collected directly in the injection plate which can then be
placed in the sequencer, without the need for an intervening
collection plate and the transfer of purified samples from the
collection plate to the injection plate. The adaptor assembly can
match a gel filtration plate with any of a variety of injection
plates of different configurations that are designed for sequencers
from different suppliers.
[0008] The components of the adaptor assembly include a support
base on which the injection plate rests and a bracket that holds
together the gel filtration plate, the injection plate, and the
support base. The bracket keeps the filtration plate and injection
plate in alignment during centrifugation, avoiding
cross-contamination between individual reaction mixtures. At the
same time, the support base protects the lower ends of the sample
receptacles in the injection plate from potential damage during
centrifugation. These and other features, objects and advantages of
the invention will be apparent from the description that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is an exploded view in perspective of an adaptor
assembly in accordance with the present invention together with an
injection plate for a nucleic acid sequencer.
[0010] FIG. 2 is a cross section view of the components shown in
FIG. 1 in a fully assembled arrangement.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0011] While this invention is susceptible to a variety of
configurations, arrangements and embodiments, the following
discussion will focus on a specific example, the structural and
functional aspects of which will serve to provide an understanding
of the invention as a whole.
[0012] The exploded assembly of parts shown in FIG. 1 includes
adaptor components to hold a gel filtration plate 11 and an
injection plate 12 together and in proper alignment for
simultaneous centrifugation. Each of these two plates has an array
of 96 (8.times.12) wells as in the standard MICROTITER.RTM. (Dynex
Technologies) plates, although the invention is likewise applicable
to plates that have smaller or larger numbers of wells. Since the
wells in the filtration and injection plates shown in FIG. 1 are of
a standard spacing, the wells of one plate are readily aligned with
those of the other. Nevertheless, since the filtration plate and
the injection plate are not designed for use in the same
instrument, these plates are not themselves equipped with features
that facilitate and maintain their alignment. This function is
served instead by the adaptor components of the present invention,
which include the support base 13 and a bracket 14 which has
upwardly extending tabs 15, 16. A handle 17 is included for
attachment to the bracket 14 to allow the user to lift the entire
assembly and transport the assembly from the centrifuge to the
sequencer.
[0013] The filtration plate 11 is an array of flow-through wells
21, i.e., open at both the top and bottom but retaining a gel
filtration matrix in each well to absorb impurities including
unreacted dye terminators. The injection plate 12 is an array of
receptacles 22 open at the top and closed at the bottom. The
receptacles extend downward from a flat upper surface 23. Extending
downward from the periphery of the surface 23 is a skirt 24, the
receptacles being exposed at the bottom and unprotected other than
by the skirt 24. The skirt 24 in the injection plate shown in the
Figure is a partial skirt, extending over only part of the length
of each receptacle, the ends of the receptacles extending below the
skirt. Other injection plates may have a full skirt, i.e., one that
extends the full length of the receptacles, or no skirt at all.
[0014] Each of the possible injection plate configurations (i.e.,
partial skirt, full skirt, or no skirt) is accommodated by the
support base 13 which supports the injection plate and fully
protects the receptacles. The support base 13 is hollow and
contains a peripheral wall 25 that is of sufficient height to cover
the protruding ends of the receptacles in the injection plate. In
the example shown in the Figure, the support base 25 fits inside
the skirt 24 of the injection plate. Apertures 26 in the support
base are arranged in the same 8.times.12 array as the flow-through
wells 21 in the filtration plate and the receptacles 22 in the
injection plate, to receive the closed ends of the receptacles.
[0015] The bracket 14 has a flat bottom and side walls 27 on all
four sides. The bottom has openings 28 to provide lightweight
construction, and upwardly extending tabs 15, 16 on opposing side
walls. The side walls are spaced widely enough to receive the
entire support base 13 and to thereby secure the support base in a
fixed position relative to the bracket. By engaging the receptacles
in the injection plate 12, the support base 13 also stabilizes the
position of the injection plate 12 relative to the bracket 14. The
tabs 15, 16 extend upward to contact the side edges of the
injection plate 12.
[0016] At the upper end of each tab is a small hole or eye (only
one of which 32 is visible in the Figure). The two eyes receive
hooks 33, 34 which are at the ends of the handle 17. The handle 17
is a U-shaped rod extending over the filtration plate to be grasped
by the user's hand.
[0017] The components of the adaptor assembly are shown fully
assembled in the cross section view of FIG. 2. The wells 21 of the
filtration plate 11 are partially filled with gel matrix 22 which
is supported in each well in a manner that allows the liquid
reaction mixture to flow through the well to the receptacles 41
below.
[0018] The foregoing is offered primarily for purposes of
illustration. Further alternatives as well as modifications and
variations of the configurations, systems, materials, and
procedural steps described above, which will be apparent to those
skilled in the art upon reading this specification, are included
within the scope of this invention.
* * * * *